Overall Project The long-term objective of this Program Project is to perform a systematic analysis of the cellular and molecular mechanisms regulating thymic epithelial cell (TEC) homeostasis and the role of TECs in the involution of the adult thymus. The thymus is the primary lymphoid organ responsible for the generation of functional T cells, and is therefore critical for generation and maintenance of adaptive immunity. It is also one of the first organs to undergo significant age- related organ degeneration, termed thymic involution. Thymic involution results in a dramatic drop in the production of new T cells, and is a significant contributing factor in immune senescence. Despite the importance of this subject for human health, the molecular mechanisms that operate in the postnatal thymus and mediate thymic homeostasis and involution are largely unknown. As a result, many aspects of thymic involution are poorly understood and/or controversial. Understanding the relative contributions of the mechanisms underlying this process would have significant implications for improving human health. The proposed Program Project is designed to identify mechanisms and pathways that can be exploited for induction of thymic rebound and/or prevention of involution, focused on the role of changes in TECs. The two projects will test two central hypotheses: 1)that failure to maintain postnatal thymic epithelial cell homeostasis is a causative mechanism in thymic involution;and 2) that down regulation of Foxnl, a pivotal regulator of TEC development and maintenance, is a primary molecular mechanism in thymic involution. These hypotheses will be addressed through three major Program objectives which will investigate: a) the lineage relationships of TEC subsets in the postnatal thymus;b) the molecular mechanisms driving TEC homeostasis;and c) whether these mechanisms are causal for involution. The scientific projects share the common goal of identifying the cellular and molecular mechanisms resulting in the failure of TEC maintenance in thymic involution. The approaches used are all grounded in the central idea that the mechanisms that regulate fetal thymus development and TEC differentiation provide a conceptual framework for understanding thymic aging and regeneration. The individual projects in the P01 will take overlapping and complementary approaches to test these hypotheses that combine the unique expertise, experimental tools, and institutional resources of the investigators. 5'0 (gyp [unreadable]_o 02, '<'O -r- off- Qua? c-"[unreadable] (.c d-0 -a) (=0 o:[unreadable] CD= Q[unreadable]' O.5 -00 n[unreadable]) Erika Schwabe Assistant rants Officer Modified Project Summary/Abstract Section ... 0 Project I The cellular mechanism that maintains the postnatal thymic epithelial (TE) compartment is currently unknown. Precedent from other organs suggests two possible models: the TE may be maintained by a stem/progenitor cell mechanism, as in the intestine, or by mitosis of terminally differentiated cells, as in the liver. Some recent data have been interpreted to support a stem cell mechanism, however, the results from these studies would con be consistent with either of the above scenarios. Unequivocal discrimination between these possibilities is vii [unreadable]33m R-) can twig vin C-0 important, since development of effective strategies aimed at thymus regeneration will depend on a clear thymus is also unknown. strongly implicated in regulation of TEC differentiation, at least in fetal stages, but any role in the postnatal and in TESC/TEPC maintenance and/or function, is unknown. The Wnt signaling pathway has also been proliferation, and is widely expressed in postnatal TECs;however, its role in the postnatal thymus generally, shown that the transcription factor Foxnl is required for fetal thymic epithelial cell (TEC) differentiation and regulating the postnatal thymus are also known. In particular, the Manley and Blackburn labs and others have in the postnatal thymus. Just as candidate cell populations have been identified, molecular candidates for laboratory and others has identified several candidate cell populations as containing potential stem/progenitors understanding of the target cell population(s) for therapeutic intervention. Previous work from the Blackburn In this project, we will test the overall hypothesis that changes in the molecular mechanisms that control [unreadable]>_ coo w.:. m-[unreadable] Q.. c-0 (n3-[unreadable] [unreadable]_o M,[unreadable] (L6 w-0 t0/0 DLL a'c .0) '[unreadable]_ BCD TESC/TEPC maintenance during postnatal thymus homeostasis contribute to thymic involution, by testing overcome thymic involution. This project will thus address one of the central hypotheses of the Program approaches to evaluate the role of Foxnl and the Wnt signaling pathway in molecular regulation of specific subsets of postnatal TECs will affect TEC homeostasis and involution. These aims will use genetic whether postnatal TESC/TEPC do not express Foxnl;and determining if modulation of Wnt signaling in homeostatic maintenance of the postnatal thymic stroma and will test whether de-repressing Wnt signaling can Project, that failure to maintain postnatal TEC homeostasis is a causative mechanism in thymic involution. .-. [unreadable]<3--cv"00M[unreadable]-35*' -0o pt- 7a- ... o-0 [unreadable]'.m W[unreadable][unreadable] BCD 3-[unreadable] 3t,5 -a' C/) Modified Project Summary/Abstract Section Project 2 Thymic epithelial cells (TECs) comprise a diverse group of cells that form a complex microenvironmental network required for T cell development and repertoire selection. Several years ago, we defined four major TEC subsets in the postnatal thymus based on differential expression of distinct keratin (K) species together with the ability to bind the lectin, UEA-1. This proposal focuses on pathways and mechanisms involved in TEC subset development, homeostasis and involution, with particular emphasis on medullary epithelium. Development of a functional medullary epithelial compartment is vital for establishing central tolerance. -aoo[unreadable] 000 Q[unreadable]) o)- con -[unreadable]Q m Medullary TECs (mTECs) are unique in their expression of a highly diverse array of tissue restricted antigens and functional precise mechanisms that regulate their development and homeostasis, as well as their lineage relationships advancing age. Despite a growing appreciation for the essential role of mTECs in negative selection, the negative selection and central tolerance, thereby increasing the risk of developing autoimmune disease with that progressive loss of a previously established mTEC compartment during thymus involution compromises well established that the incidence of autoimmunity increases with age. Therefore, an attractive hypothesis is that autoimmunity develops in mice with genetic defects that prevent mTEC development during ontogeny. It is occurs in patients or mice with genetic defects in TRA expression. In addition, our lab and others have shown (TRAs) that are required to negatively select self-reactive thymocytes. Widespread autoimmune disease E 3-0 con 0-[unreadable]m relationship between the major mTEC subsets defined as K5K14UEA-1 and K5K14UEA-14. This hypothesis thymocyte differentiation. Specific Aim 3 will determine whether Stat3 activation in mTEC precursors is development in RAG-2 mice that are normally devoid of organized medullary regions due to an early block in data show that expression of a K5 promoter driven constitutively active Stat3 transgene induces mTEC identification of the molecular pathways that regulate mTEC differentiation and proliferation. Our preliminary will be tested in Aim 1. A strategy to prevent or reverse loss of the mTEC population with age requires required to generate or sustain the mTEC compartment. potential remain to be determined. Our preliminary data suggest a precursor-progeny E nom[unreadable].r ..v >-a (-0 .-. -vo C.. ago (n' ma) N-0 -oo 07m SQL ma) 7_< (C0 mama) -ow -[unreadable]Dv-n 3.0 (<p cam' (n: 0G,[unreadable] 5'c Modified Project Summary/Abstract Section Project 3 Entirety of Project 3 has been deleted. E m Core B Modified Project Summary/Abstract Section Entirety of Core B has been deleted. ,a, 0 C,'